The present invention relates to the field of cytokines, in particular to those cytokines which synergize with Interleukin-2 (IL-2) to activate cytotoxic lymphocytes.
xe2x80x98Cytokinexe2x80x99 is one term for a group of protein cell regulators, variously called lymphokines, monokines, interleukins and interferons, which are produced by a wide variety of cells in the body, play an important role in many physiological responses, are involved in the pathophysiology of a range of diseases, and have therapeutic potential. This heterogeneous group of proteins has the following characteristics in common. They are low molecular weight (xe2x89xa680 kDa) secreted proteins which are often glycosylated; are involved in immunity and inflammation where they regulate the amplitude and duration of a response; and are usually produced transiently and locally, acting in a paracrine or autocrine, rather than endocrine manner. Cytokines are extremely potent, generally acting at picomolar concentrations; and interact with high affinity cell surface receptors specific for each cytokine or cytokine group. Their cell surface binding ultimately leads to a change in the pattern of cellular RNA and protein synthesis, and to altered cell behavior. Individual cytokines have multiple overlapping cell regulatory actions.
The response of a cell to a given cytokine is dependent upon the local concentration of the cytokine, the cell type and other cell regulators to which it is concomitantly exposed. The overlapping regulatory actions of these structurally unrelated proteins binding to different cell surface receptors is at least partially accounted for by the induction of common proteins which can have common response elements in their DNA. Cytokines interact in a network by: first, inducing each other; second, transmodulating cytokine cell surface receptors and third, by synergistic, additive or antagonistic interactions on cell function. [Immunology Today 10 No. 9 p 299 (1989)].
The potential utility of cytokines in the treatment of neoplasia and as immunoenhancing agents has recently been demonstrated in studies using human recombinant interleukin-2 (rIL-2). Natural Interleukin-2 (IL-2) is a lymphokine which is produced and secreted by T-lymphocytes. This glycoprotein molecule is intimately involved in the induction of virtually all immune responses in which T-cells play a role. B cell responses in vitro are also enhanced by the presence of IL-2, and IL-2 has also been implicated as a differentiation inducing factor in the control of B and T lymphocyte responses.
Administration of human rIL-2 has been shown in some cases to result in regression of established tumors in both experimental animals [J. Exp. Med 161:1169-1188, (1985)] and in man [N. Engl. J. Med 313: 1485-1492, (1985) and N. Engl. J. Med 316:889-897 (1987)]. The anti-tumor effects of rIL-2 are thought to be mediated by host cytotoxic effector lymphocytes which are activated by rIL-2 in vivo [J. Immunol. 139:285-294 (1987)]. In addition, results from animal models suggest that rIL-2 might also have value in the treatment of certain infectious diseases [J. Immunol. 135:4160-4163 (1985) and J. Virol. 61:2120-2127 (1987)] and in ameliorating chemotherapy-induced immunosuppression [Immunol Lett. 10:307-314 (1985)].
However, the clinical use of rIL-2 has been complicated by the serious side effects which it may cause [N. Engl. J. Med. 313:1485-1492 (1985) and N. Engl. J. Med. 316:889-897 (1987)]. One approach to improving the efficacy of cytokine therapy while reducing toxicity is to use two or more cytokines in combination. For example, synergistic antitumor activity has been shown to result when rIL-2 is administered to tumor-bearing mice together with recombinant interferon alpha (rIFN alpha) [Cancer Res. 48:260-264 (1988) and Cancer Res. 48:5810-5817 (1988)] or with recombinant tumor necrosis factor alpha (rTNF alpha)[Cancer Res. 47:3948-3953 (1987)]. Since the antitumor effects of IL2 are thought to be mediated by host cytotoxic effector lymphocytes, it would be of interest to identify and isolate novel cytokines which synergize with rIL2 to activate cytotoxic lymphocytes in vitro. These novel cytokines would also be useful as antitumor agents when administered in combination with rIL2 in vivo.
The present invention is a novel cytokine protein called Cytotoxic Lymphocyte Maturation Factor (CLMF) also called IL-12 which is produced and synthesized by cells capable of secreting CLMF such as mammalian cells particularly human NC-37 B lymphoblastoid cells (ATCC CCL 214 American Type Culture Collection, Rockville, Md.). CLMF synergistically induces with low concentrations of IL-2 (IL-2) the cytolytic activity of Lymphokine Activated Killer (LAK) cells, and CLMF is capable of stimulating T-cell growth.
The present invention is directed toward the process of isolating CLMF in a substantially pure form.
The process comprises the following:
stimulating NC-37 B lymphoblastoid cells to produce and secrete cytokines into a supernatant liquid;
collecting the supernatant liquid produced by the stimulated cells;
separating the supernatant liquid into protein fractions;
testing each protein fraction for the presence of CLMF;
retaining the protein fractions which are able to stimulate T-cell growth, said fractions containing an active protein which is responsible for the T-cell stimulating activity of the protein fractions;
isolating said active protein into a substantially pure form, said protein being Cytolytic Lymphocyte Maturation Factor (CLMF). CLMF is a 75 kilodalton (kDa) heterodimer comprised of two polypeptide subunits, a 40 kDa subunit and a 35 kDa subunit, which are bonded together via one or more disulfide bonds.
The process of this invention is capable of purifying CLMF from any liquid or fluid which contains CLMF together with other proteins. Also claimed are the protein fractions capable of stimulating T-cell growth, the substantially purified active protein, CLMF, obtained from the above described process, the isolated cloned genes encoding the 40 kDa subunit and the 35 kDa subunit, vectors containing these genes and host cells transformed with the vectors containing the genes.
In addition a method for stimulating LAK cells and T-cells comprised of treating these cells with CLMF alone or with IL-2 is claimed. Also claimed are isolated antibodies capable of binding to CLMF.
Antibodies to CLMF
Monoclonal antibodies prepared against a partially purified preparation of CLMF have been identified and characterized by 1: immunoprecipitation of 125I-labelled CLMF, 2: immunodepletion of CLMF bioactivity, 3: western blotting of CLMF, 4: inhibition of 125I-CLMF binding to its cellular receptor and 5: neutralization of CLMF bioactivity. Twenty hybridomas were identified which secreted anti-CLMF antibodies. The antibodies immunoprecipitate 125I-labelled CLMF and immunodeplete CLMF bioactivity as assessed in the T-cell proliferation and LAK cell induction assays. Western blot analysis demonstrate that each antibody binds to the 70 kDa heterodimer and to the 40 kDa subunit. These data demonstrated that the 20 antibodies were specific for CLMF and in particular for the 40 kDa subunit of CLMF. A CLMF receptor binding assay has been developed to evaluate the ability of individual antibodies to inhibit CLMF binding to its cellular receptor. The assay measures the binding of 125I-labelled CLMF to PHA activated PBL blast cells in the presence and absence of each antibody. Of the 20 antibodies tested, 12 antibodies inhibit greater than 60% of the 125I-labelled CLMF binding to the blast cells. Two inhibitory antibodies, 7B2 and 4A1, neutralize CLMF bioactivity while one non-inhibitory antibody, 8E3, does not neutralize CLMF bioactivity. These data confirm that antibodies which block 125I-labelled CLMF binding to its cellular receptor will neutralize CLMF bioactivity as assessed by the T-cell proliferation and LAK cell induction assays. The ability of the antibodies specific for the 40 kDa subunit of CLMF to neutralize CLMF bioactivity indicates that determinants on the 40 kDa subunit are necessary for binding to the CLMF cellular receptor.
Utility for the Monoclonal Anti-Human CLMF Antibodies
The monoclonal anti-CLMF antibodies provide powerful analytical, diagnostic and therapeutic reagents for the immunoaffinity purification of natural and recombinant human CLMF, the development of human CLMF immunoassays, the identification of the active site of the 40 kDa subunit of CLMF and as possible therapeutic treatments which require selective immunosuppression of cytotoxic T cells, such as in transplantation. Monoclonal antibodies which recognize different epitopes on human CLMF can be used as reagents in a sensitive two-site immunoassay to measure levels of CLMF in biological fluids, cell culture supernatants and human cell extracts.
The present invention is directed to monoclonal antibodies against CLMF which exhibit a number of utilities including but not limited to:
1. Utilizing the monoclonal antibodies as affinity reagents for the purification of natural and recombinant human CLMF;
2. Utilizing the monoclonal antibodies as reagents to configure enzyme-immunoassays and radioimmunoassays to measure natural and recombinant CLMF in biological fluids, cell culture supernatants, cell extracts and on plasma membranes of human cells and as reagents for a drug screening assay;
3. Utilizing the monoclonal antibodies as reagents to construct sensitive two-site immunoassays to measure CLMF in biological fluids, cell culture supernatants and human cell extracts;
4. Utilizing the monoclonal antibodies as reagents to identify determinants of the 40 kDa subunit which participate in binding to the 35 kDa subunit and which participate in binding to the CLMF cellular receptor;
5. Utilizing the intact IgG molecules, the Fab fragments or the humanized IgG molecules of the inhibitory monoclonal antibodies as therapeutic drugs for the selective blockade of proliferation and activation of cytotoxic T cells, such as in transplantation.